In the field of molecular diagnostics, the amplification and detection/quantitation of nucleic acids is of considerable significance and importance. Examples for diagnostic applications of nucleic acid amplification and detection are for the detection and amplification of microbial nucleic acids. Such microbial nucleic acids may include bacterial nucleic acids and/or viral nucleic acids. The amplification and detection/quantitation techniques are suitable for viral nucleic acid targets, such as Human Papilloma Virus (HPV) or West Nile Virus (WNV), or the routine screening of blood donations for the presence of Human Immunodeficiency Virus (HIV), Hepatitis A Virus (HAV), Hepatitis B Virus (HBV), or Hepatitis C Virus (HCV). The amplification and detection/quantitation techniques are also suitable for bacterial nucleic acid targets or the analysis of oncology markers or the like.
The most prominent and widely used method for amplification (and detection/quantitation) of nucleic acid targets is the Polymerase Chain Reaction (PCR). Other amplification techniques include Ligase Chain Reaction, Polymerase Ligase Chain Reaction, Gap-LCR, Repair Chain Reaction, 3 SR, NASBA, Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA), and Qβ-amplification.
Automated systems for PCR-based analysis often make use of a real-time detection of product amplification during the PCR process in the same reaction vessel. Key to such methods is the use of modified oligonucleotides carrying reporter groups or labels. PCR utilizes a polymerase enzyme (U.S. Pat. Nos. 4,683,195 and 4,683,202). Related significant improvements are, e.g., real-time detection of amplified products during PCR utilizing modified oligonucleotides carrying reporter groups or labels known as hydrolization or 5′-nuclease probes such as used in commercial assays on COBAS® TaqMan® (U.S. Pat. Nos. 5,210,015 and 5,487,972). Other improved amplification and detection methods are known as Molecular Beacons technology (International Patent Publication No. WO 95/13399) or methods utilizing an oligonucleotide comprising a minor groove binder (MGB) portion (International Patent Publication Nos. WO 03/062445 and WO 2006/135765). It is further known that the use of primers containing an added oligonucleotide with a high GC content at the 5′ terminus of at least one of these primers displays an improvement in amplification efficiency (Liu, et al., Genome Research 7:389-398 (1997); International Patent Publication No. WO 01/94638; and U.S. Patent Publication No. US 2004/0110182). The final quantity of the amplified product after approximately 12 to 40 cycles of PCR is markedly higher for primers to which, e.g., a GGAC unit has been added to the 5′ termini than for the unmodified primers.
Afonina, et al., BioTechniques 43(3):1-3 (2007); International Patent Publication No. WO 2006/135765) describe the increase of real-time PCR fluorescent signal and thereby obtaining improved amplification efficiency by using primers with short adenine and thymine rich flaps, scattered randomly, at the 5′ terminus and minor groove binder (MGB) fluorescent hybridization probes.
Similarly, Babiel, et al. (U.S. Pat. No. 9,447,476) describe how the addition of polyN to primers can result in the reduction or suppression of the formation of unwanted high molecular weight products, thereby avoiding false-negative or false-positive results.
Thus, there is always a need in the art for improvements on existing methods. For example, there is a need in the art to provide a method for simple and reliable detection and quantitation of a nucleic acid target. There is, in particular a need for improving the efficiency of DNA amplification and detection/quantitation.